Spink1 targeted therapy

ABSTRACT

The present invention relates to compositions and methods for cancer therapy, including but not limited to, targeted inhibition of cancer markers. In particular, the present invention relates to SPINK1 as a clinical target for prostate cancer.

This application claims priority to provisional application 61/306,267,filed Feb. 19, 2010, which is herein incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant numbersCA069568, CA132874 and CA111275 awarded by the National Institutes ofHealth and W81XWH-08-1-003 awarded by the Army Medical Research andMaterial Command. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for cancertherapy, including but not limited to, targeted inhibition of cancermarkers. In particular, the present invention relates to SPINK1 as aclinical target for prostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common nondermatologic cancer and the secondmost common cause of cancer-related deaths in American men. The numberof prostate cancers recorded in cancer registries in the United Statesand the United Kingdom has increased markedly in the past 15 years. Thischange predominantly represents an increase in the number of cancersdiagnosed rather than a real increase in the number of cancers in thepopulation. In 2006, 234,460 new cases and 27,350 deaths were estimatedto occur. It was determined that approximately 91% of these new caseswould be diagnosed at local or regional stages.

Prostate cancer (PCa) is typically diagnosed with a digital rectal examand/or prostate specific antigen (PSA) screening. An elevated serum PSAlevel can indicate the presence of PCa. PSA is used as a marker forprostate cancer because it is secreted only by prostate cells. A healthyprostate will produce a stable amount—typically below 4 nanograms permilliliter, or a PSA reading of “4” or less—whereas cancer cells produceescalating amounts that correspond with the severity of the cancer. Alevel between 4 and 10 may raise a doctor's suspicion that a patient hasprostate cancer, while amounts above 50 may show that the tumor hasspread elsewhere in the body.

When PSA or digital tests indicate a strong likelihood that cancer ispresent, a transrectal ultrasound (TRUS) is used to map the prostate andshow any suspicious areas. Biopsies of various sectors of the prostateare used to determine if prostate cancer is present. Treatment optionsdepend on the stage of the cancer. Men with a 10-year life expectancy orless who have a low Gleason number and whose tumor has not spread beyondthe prostate are often treated with watchful waiting (no treatment).Treatment options for more aggressive cancers include surgicaltreatments, such as radical prostatectomy (RP) in which the prostate iscompletely removed (with or without nerve sparing techniques), andradiation, applied through an external beam that directs the dose to theprostate from outside the body or via low-dose radioactive seeds thatare implanted within the prostate to kill cancer cells locally.Anti-androgen hormone therapy is also used, alone or in conjunction withsurgery or radiation. Hormone therapy uses luteinizing hormone-releasinghormones (LH-RH) analogs, which block the pituitary from producinghormones that stimulate testosterone production. Patients must haveinjections of LH-RH analogs for the rest of their lives.

While surgical and hormonal treatments are often effective for localizedPCa, advanced disease remains essentially incurable. Androgen ablationis the most common therapy for advanced PCa, leading to massiveapoptosis of androgen-dependent malignant cells and temporary tumorregression. In most cases, however, the tumor reemerges with a vengeanceand can proliferate independent of androgen signals.

Thus, additional therapies that target prostate cancers are needed.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for cancertherapy, including but not limited to, targeted inhibition of cancermarkers. In particular, the present invention relates to SPINK1 as aclinical target for prostate cancer.

For example, in some embodiments, the present invention provides amethod of inhibiting at least one biological function of SPINK1 (e.g.,SPINK secreted by a cell) comprising contacting the SPINK1 with areagent that inhabits at least one biological function of the SPINK1. Insome embodiments, the method comprises contacting a SPINK1 polypeptidewith an antibody that specifically binds to the SPINK1 polypeptide andinhibits at least one biological function of the SPINK1 polypeptide(e.g., those described herein). In some embodiments, the cell is acancer cell (e.g., a prostate cancer cell). In some embodiments, themethod comprises administering a nucleic acid based therapeutic (e.g.,siRNA, antisense and the like) that inhibits expression of a SPINK1 mRNAor polypeptide. In some embodiments, the cell is in vivo, in vitro, exvivo, or in an animal (e.g., a human or a non-human mammal). In someembodiments, the cell does not harbor an ETS gene fusion (e.g., aTMPRSS2:ETS fusion). In some embodiments, the reagent inhibits theproliferation or the invasiveness of the cell. In some embodiments, oneor more chemotherapeutic agents are administered in combination with theantibody.

Further embodiments provide a method of inhibiting at least onebiological function of EGFR, for example, alone or in combination withinhibition of a biological function of SPINK. In some embodiments, ansiRNA reagent, antisense reagent or a monoclonal antibody is used toinhibit a biological function of EGFR.

In additional embodiments, the present invention provides a kit,comprising a pharmaceutical composition that inhibits at least onebiological function of SPINK1 and/or EGFR (e.g., SPINK secreted by acell, wherein the cell is located in vivo, ex vivo, in vitro or in ananimal). In some embodiments, the composition comprises a nucleic acidbased therapeutic (e.g., siRNA, antisense or the like) or an antibody(e.g. an antibody that specifically binds to SPINK1 and inhibits atleast one biological function of SPINK1).

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows that SPINK1 promotes cell proliferation and invasion inSPINK1 negative cell lines. (A) SPINK1 stimulates cell proliferation inSPINK1⁻/ETS⁻ cell lines. (B) SPINK1 mediates invasion of benignimmortalized prostate cell line RWPE as measured by Boyden chamberMatrigel invasion assay. (C) 22RV1 cells transfected with siRNA againstSPINK1 showed decrease in cell invasion.

FIG. 2 shows that stable knockdown of SPINK1 inhibits cell proliferationand invasion of SPINK1⁺ prostate cancer cells. (A) SPINK1 knockdown in22RV1 cells was confirmed at the transcript level by quantitative PCRand protein level by immunofluorescence (upper insets; 600×magnification) using an antibody against SPINK1. (B) Boyden chamberMatrigel invasion assay demonstrates decrease in cell invasion in stablepooled shSPINK1 knockdown 22RV1 cells as compared to pooled shNS vectorcells. (C) Cell proliferation assay showing decrease in proliferation inpooled or single clone shSPINK1 knockdown 22RV1 cells as compared topooled shNS vector cells at the indicated time points. (D) Soft agarcolony assay showed decrease in the number of colonies in stable pooledshSPINK1 knockdown 22RV1 cells as compared to pooled shNS vector cells.

FIG. 3 shows that Anti-SPINK1 mAb attenuates in vitro proliferation andinvasion exclusively in SPINK1+/ETS-prostate cancer cells. (A) Cellproliferation of DU145, PC3 and 22RV 1 cells was assessed in thepresence of 1 μg/ml SPINK1 mAb or IgG mAb (B) As in A except using 22RV1cells and 0.5-1 μg/ml SPINK1 mAb or IgG mAb. (C) Effect of SPINK1 mAb orIgG mAb on invasion of SPINK1+/ETS-cells (22RV 1 and CWR22PC) andSPINK1+/ETS-cells (DU145, PC3, LNCaP and VCaP).

FIG. 4 shows SPINK1 as a therapeutic target in SPINK1⁺ prostate cancer.(A) Chick chorioallantoic membrane (CAM) assay quantifying intravasatedRWPE cells upon stimulation with rSPINK1 (n=6 in each group). (B) CAMassay using 22RV1 cells in the presence of IgG mAb, SPINK1 mAb or C225(n=5 in each group), with fold change of intravasated cells compared toIgG mAb plotted. (C) As in B, except using PC3 cells. (D) Subcutaneousxenograft growth of shNSluciferase (luc) or shSPINK1-luc 22RV1 cellsimplanted in male BALB/C nu/nu mice (n=10 in each group). (E) As in D,except using 22RV1-luc cell xenografts treated with control IgG mAb(n=8), SPINK1 mAb (n=6) or C225 (n=8) (10 mg/kg body weight) twice aweek. (F) Same as in E, except mice (n=7 per group) were treated with acombination of SPINK1 mAb and C225 mAb (10 mg/kg body weight for both).(G) As in E & F, except using PC3-luc xenografts treated with controlIgG mAb, SPINK1 mAb or C225 (n=8 per group) (10 mg/kg body weight) aloneor in combination twice a week. (H) Representative bioluminescenceimages from mice in D bearing pooled shNS-luc or shSPINK1-luc xenograftsand % reduction in tumor volume at week 5. (I) Same as H, exceptbioluminescence images from mice bearing 22RV1-luc xenografts in (toppanel) or PC3-luc (lower panel) mice treated with IgG mAb, SPINK1 mAb,or C225 mAb alone or in combination, with comparative % reduction plotin tumor volume at week 5.

FIG. 5 shows bacterial expression vectors (pQE-9) constructed to produceN-6× His-tag SPINK1 recombinant protein (rSPINK1) from human cDNA.

FIG. 6 shows the effect of conditioned medium (CM) collected from 22RV1cells (10 kd fraction) or rSPINK1 protein in the presence or absence ofanti-SPINK1 monoclonal antibody on breast cancer MCF7 cells invasion inBoyden chamber Matrigel invasion assay.

FIG. 7 shows the effect of conditioned medium (CM) collected from RWPEcells (10 kd fraction), multiple tag protein (including 6× His) orrSPINK1 protein on 22RV1 or RWPE cells invasion in Boyden chamberMatrigel invasion assay.

FIG. 8 shows that SPINK1 mAb reduces SPINK1+ cell motility and SPINK1knockdown alters MAPK pathway. A, Cell motility assay was carried out byplating 22RV1 cells in the presence or absence of the SPINK1 mAb or IgGmAb on a lawn of microscopic fluorescent beads on collagen coated96-well plates. B, Quantitative RT-PCR showing decrease in EGFRexpression in the 22RV1 cells. C, Western blot showing pMEK, pERK, pAKT,tMEK, tERK and tAKT expression levels in shNS and shSPINK1 22RV1 cells(single clone). D, Same as C, except pERK and tERK levels in the 22RV1cells treated with IgG or SPINK1 mAb. pMEK, pERK and pAKT denotesphosphorylated-MEK, -ERK or -AKT and tMEK, tERK or tAKT denotestotal-MEK, -ERK or -AKT levels.

FIG. 9 shows that SPINK1 mAb induces decrease in tumor proliferationindex, but has no effect on toxicity markers. A, Ki-67immunohistochemical (IHC) staining of tumor tissue showing Ki-67positive nuclei in SPINK1 mAb treatment group as compared to controlIgG. B, Pancreatic toxicity markers showing amylase and lipase levels(U/L) in the serum samples collected from control IgG or anti-SPINK1 mAbtreated mice. C, Same as B, except hepatic toxicity markers showingalkaline phosphatase (ALKP), alanine aminotranferease (ALT) andaspartate aminotransferase (AST) levels (U/L). D, Same as B, exceptgeneral health profile markers showing CK: creatinine kinase (U/L);CHOL: cholesterol; TRIG: triglycerides; CREA: creatine; Ca: calcium; Mg:magnesium; PHOS: phosphorus (mg/ml).

FIG. 10 shows that Anti-IgG or -SPINK1 mAb or C225 administration has noeffect on mouse body weight. A, Body weight was recorded for the micetreated with control IgG or anti-SPINK1 monoclonal antibodies. B, sameas A except mice were treated with a combination of SPINK1 monoclonalantibody and C225. C, same as A except mice were xenografted with PC3cells.

FIG. 11 shows that SPINK1 mediates its oncogenic effects in part throughEGFR. (A) Immunoprecipitation using anti-IgG, anti-SPINK1 or anti-GST ofexogenous SPINK1-GST, GST or GST-VEGFR added to HEK-293 cellstransfected with EGFR and immunoblotted with anti-EGFR (top panel), andimmunoprecipitation using anti-IgG or anti-SPINK1 of exogenousSPINK1-GST added to 22RV1 cells and immunoblotted with anti-EGFR (bottompanel). (B) Western blot showing EGFR phosphorylation in response torSPINK1 (100 ng/ml) or EGF (10 ng/ml) stimulation. (C) Invasion assayshowing siRNA mediated EGFR knockdown 22RV1 cells treated with 10 ng/mlof rSPINK1 (D) Same as in C, except with RWPE cells. (E) Invasion assayshowing rSPINK1 (10 ng/ml) stimulated RWPE cells in the presence orabsence of C225 (cetuximab, 50 μg/ml) or IgG mAb (50 μg/ml) (F) Invasionassay showing the effect of IgG or C225 antibody on SPINK1+ and SPINK1−cancer cells. (G) As in F, except 22RV1 cells were treated with acombination of anti-SPINK1 and/or C225 mAb (1 μg/ml and 50 μg/mlrespectively). (H) Cell proliferation assay using the indicated cells inthe presence of IgG mAb or C225.

FIG. 12 shows expression of SPINK1 in a prostate cell line panel byquantitative RT-PCR.

FIG. 13 shows that CM collected from 22RV1 cells induces cell invasion,but not CM from LNCaP cells.

FIG. 14 shows that PRSS1 (trypsin1) knockdown in 22RV1 cells has noeffect on SPINK1 mediated cell invasion. A, Expression of PRSS1(trypsin1) by qPCR. Pancreatic cancer cells, CAPAN1 was used as acontrol. B, Same as A except SPINK1 was knocked down in the 22RV1 cellline using siRNA against SPINK1. C, Western blot showing trypsin levelsin the 22RV1 cells stimulated with rSPINK1 or EGF at different timepoints as indicated. D-E, PRSS1 was knocked down in the 22RV1 cell lineusing multiple siRNA constructs. D, PRSS1 expression was determined byqPCR and E, the effect on invasion was determined by Boyden chamberMatrigel invasion assay.

FIG. 15 shows that exogenous rSPINK1 has no effect on PSA in 22RV1cells. A, Western blot showing no change in PSA level in 22RV1 cell linestimulated with rSPINK1 (100 ng/ml) or EGF (10 ng/ml). B, Matrigelinvasion assay using 22RV1 cell line in the presence of IgG or PSAmonoclonal antibody.

FIG. 16 show that exogenous SPINK1 induces EGFR dimerization andphosphorylation. A, Western blot showing EGFR phosphorylation in thestable shNS, shSPINK1 pool and in single shSPINK1 clone. B, Non-reducingWestern blot showing EGFR dimerization after stimulating with rSPINK1(100 ng/ml) and EGF (10 ng/ml) as indicated in the presence or absenceof crosslinking reagent BS3 (3 mM in PBS).

FIG. 17 shows mapping of the epitope on the SPINK1 monoclonal antibody.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “inhibits at least one biological activity ofserine peptidase inhibitor, Kasal type I (SPINK1)” refers to any agentthat decreases any activity of SPINK1 (e.g., including, but not limitedto, the activities described herein), via directly contacting SPINK1protein, contacting SPINK1 mRNA or genomic DNA, causing conformationalchanges of SPINK1 polypeptides, decreasing SPINK1 protein levels, orinterfering with SPINK1 interactions with signaling partners, andaffecting the expression of SPINK1 target genes. Inhibitors also includemolecules that indirectly regulate SPINK1 biological activity byintercepting upstream signaling molecules.

As used herein, the term “siRNAs” refers to small interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to, or substantially complementary to, a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand;” the strand homologous to the target RNA molecule isthe “sense strand,” and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the term “outlier expression of SPINK1” refers to analtered level of expression of SPINK1 nucleic acid (e.g., mRNA) orprotein relative to the level normally found (e.g., the level in asubject not diagnosed with cancer). In some embodiments, normal levelsare the average level in a population of one or more individuals notdiagnosed with cancer. In other embodiments, normal levels aredetermined within other tissues of the individual to be diagnosed. Insome embodiments, expression is altered by at least 10%, preferably atleast 20%, even more preferably at least 50%, yet more preferably atleast 75%, still more preferably at least 90%, and most preferably atleast 100% relative to the level of expression normally found (e.g., innon-cancerous tissue). Expression levels may be determined using anysuitable method. In some embodiments, samples positive for outlierexpression of SPINK1 are those whose expression differs by greater thanabout 0.1, preferably greater than 0.2, and even more preferably greaterthan 0.5 normalized expression units. Normalized expression units may becalculated using any suitable method.

As used herein, the term “overexpression of SPINK1” refers to a higherlevel of expression of SPINK1 nucleic acid (e.g., mRNA) or proteinrelative to the level normally found. In some embodiments, expression isincreased at least 10%, preferably at least 20%, even more preferably atleast 50%, yet more preferably at least 75%, still more preferably atleast 90%, and most preferably at least 100% relative to the level ofexpression normally found. The level of expression normally found may bedetermined using any number of suitable parameters. Examples include,but are not limited to, the level in non-cancerous prostate (e.g., anaverage of the level of SPINK1 expression in prostate tissues frommultiple subjects not diagnosed with prostate cancer), the level innon-cancerous tissues (e.g., an average of the level of SPINK1expression in non-prostate tissues from multiple subjects not diagnosedwith cancer), the level in non-cancerous prostate cell lines, or arelative level of expression (e.g., the level over time in the sameindividual). Expression levels may be determined using any suitablemethod, including, but not limited to, those disclosed herein. In someembodiments, expression levels are compared to the level of expressionof a known gene (e.g., the level of expression or the relativeexpression). In some embodiments, the known gene is PSA. As used herein,the term “gene expression associated with prostate cancer recurrence”refers to a gene expression profile (e.g., outlier expression of SPINK1)associated with prostate cancer recurrence (e.g., in the prostate ormetastatic) following treatment (e.g., surgery) for a primary tumor. Insome embodiments, prostate cancer recurrence is increased at least 10%,preferably at least 20%, even more preferably at least 50%, yet morepreferably at least 75%, still more preferably at least 90%, and mostpreferably at least 100% relative to the level of recurrence inrepresentative subject population (e.g., average of a large population(e.g., one or more, preferably 100 or more, even more preferably 1000 ormore and still more preferably 10,000 or more subjects) of subjectslacking “outlier expression of SPINK1”).

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular antibody.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is,the native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods described herein.In some embodiments, test compounds include antisense compounds.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the describedcompositions and methods.

As used herein, the term “prostate sample” refers to any samplecontaining prostate cells or secretions. Example of prostate samplesinclude, but are not limited to, a prostate tissue sample (e.g., abiopsy sample) or a urine sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for cancertherapy, including but not limited to, targeted inhibition of cancermarkers. In particular, the present invention relates to SPINK1 as aclinical target for prostate cancer.

When applied to the Oncomine database (Rhodes et al., Proc Natl Acad SciUSA 101, 9309 [2004]; Rhodes et al., Neoplasia 6, 1 [2004]), themethodology termed Cancer Outlier Profile Analysis (COPA) correctlyidentified several known oncogenes, including PBX1 in leukemia and CCND1in multiple myeloma (Tomlins et al., Science 310, 644 [2005]). Inaddition, COPA nominated ETS family genes as candidate oncogenes inprostate cancer prompting the discovery of recurrent chromosomalrearrangements involving ERG or ETV1 and the androgen-regulated geneTMPRSS2 (Tomlins et al., [2005], supra).

As 50-70% of prostate cancers harbor TMPRSS2:ETS fusions, experimentswere conducted to identify additional candidate oncogenes in prostatecancers. Experiments conducted used a meta-analysis of COPA applied to 7prostate cancer profiling studies and analyzed candidates for outlierexpression in prostate cancer and mutually exclusive over-expressionwith ERG and ETV1. SPINK1, which was identified as the 2nd rankedmeta-outlier, met both criteria across 8 data sets. SPINK1 showed markedoverexpression in 50 of 325 (15.4%) profiled prostate cancers, but only1 of 56 (1.8%) benign prostate tissue samples. In all 325 profiledprostate cancer samples, SPINK1, ERG and ETV 1 showed mutually exclusiveoutlier expression. The over-expression of SPINK1 in a fraction ofcancer samples compared to benign prostate tissue and the mutuallyexclusive over-expression of SPINK1, ERG and ETV1 was confirmed byquantitative PCR. Fluorescence in situ hybridization from tissue fromone of the localized prostate cancers over-expressing SPINK1 did notreveal gene rearrangements or amplification, indicating that SPINK1 isup-regulated through increased transcription. Together these results,consistent across different assays, microarray platforms, laboratoriesand sample cohorts, demonstrate that SPINK1 is exclusivelyover-expressed in prostate cancers without TMPRSS2:ETS SPINK1s (asindicated by ERG or ETV1 over-expression).

The cDNA sequence of SPINK1 (serine peptidase inhibitor, Kazal type 1)is provided in Genbank accession number NM_(—)003122.2. The peptideencoded by SPINK1, also known as PSTI or TATI, was originally isolatedfrom bovine pancreas and human pancreatic juice; its normal function isthought to be the inhibition of trypsin in the pancreas (Haverback etal., Am J Med 29, 421-33 (1960); Kazal et al., Journal of the AmericanChemical Society 70, 3034-3040 (1948); Paju et al., Crit Rev Clin LabSci 43, 103-42 (2006); Greene et al., Methods Enzymol 45, 813-25(1976)). SPINK1 mRNA and protein have been detected in a variety ofbenign and cancerous tissues, however its expression in prostate has notbeen described (reviewed in Paju and Stenman, Crit Rev Clin Lab Sci 43,103-42 (2006), Stenman, Clin Chem 48, 1206-9 (2002)). SPINK1 encodes a79 amino acid peptide with a 23 amino acid signal peptide and isdetectable in the urine and serum of healthy individuals (Paju andStenman, supra). In addition to being strongly elevated during severeinflammation and pancreatitis, serum levels of SPINK1 may bedysregulated in numerous cancers, including pancreatic, gastric, liver,lung, breast, bladder, renal, head and neck, colorectal, kidney andovarian cancer (reviewed in Paju and Stenman, supra, Stenman, supra).

The mouse homologue (SPINK3), plays important roles in proliferation anddifferentiation of various cell types during embryonic development (Wanget al., Histochem Cell Biol 130, 387-397 (2008)). Apart from its normalexpression in pancreatic acinar cells, SPINK1 mRNA or protein is oftenexpressed in various types of human cancers (Kelloniemi et al., Urology62, 249-253 (2003); Lukkonen et al., Int J Cancer 83, 486-490 (1999);Haglund et al., Br J Cancer 54, 297-303 (1986); Higashiyama et al., Br JCancer 62, 954-958 (1990); Huhtala et al., Int J Cancer 31, 711-714(1983); Paju et al., Clin Cancer Res 10, 4761-4768 (2004); Ohmachi etal., Int J Cancer 55, 728-734 (1993)), and increased serum SPINK1 levelcorrelates with poor prognosis in some studies (Kelloniemi et al.,supra; Lukkonen et al., supra; Paju et al., supra). The prostate glandalso secretes a variety of serine proteases, most notably the kallikreinenzyme PSA, but also trypsin, which is over-expressed in prostate cancer(Bjartell et al., Prostate 64, 29-39 (2005)).

Therapies targeted against specific molecular alterations present onlyin cancer cells have revolutionized treatment in several cancers, suchas imatinib (Gleevec), which targets the BCR-ABL chimeric protein inchronic myelogenous leukemia and trastuzumab (Herceptin) targetingERBB2, which is amplified in ˜25% of breast cancers. In prostate cancer,although multiple currently approved therapies (and newer agents in latestage development) target the androgen signaling axis, additionaltargeted therapies are lacking SPINK1 encodes a cell surfaceanti-proteinase, which may be amendable to therapeutic targeting bytraditional strategies, such as monoclonal antibodies.

Experiments conducted during the course of development of embodiments ofthe present invention demonstrated that SPINK1 promotes prostate cancerproliferation and invasion through autocrine and paracrine signaling.Mutation of SPINK1 at leucine 18 (L18) in the trypsin interaction sitereduced tumor growth, angiogenesis and lung metastases in HT-29 5M21human colon carcinoma tumor xenografts, indicating that the cancerrelated phenotypes of SPINK1 may be related to its anti-proteinaseactivity (Gouger et al., Oncogene 27, 4024-4033 (2008)). Additionally,SPINK1 has been shown to engage the EGFR/mitogen-activated proteinkinase cascade in NIH3T3 fibroblasts and pancreatic cancer cells (Ozakiet al., Mol Cancer Res 7, 1572-1581 (2009)). SPINK1 was also discoveredas an apoptosis inhibitor preventing the serine protease dependent cellapoptosis of malignant cells (Lu et al., Apoptosis 13, 483-494 (2008)).The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, it is contemplated that this finding incorroboration with the experiments described herein indicates thatSPINK1 not only acts as pro-proliferation and pro-invasive autocrine,but can also make cancer cells resistant to apoptosis and acts as animportant factor in cancer progression.

Discovery of hormone-driven expression of the ERG after fusion withTMPRSS2 (Tomlins et al., Science 310, 644-648 (2005)), allows for thetreatment of androgen driven ETS-positive patients with anti-androgenagents such as abiraterone acetate or CYP17 specific inhibitor, which isknown to ablate the synthesis of androgens and estrogens that driveTMPRSS2-ERG fusions (Setlur et al., J Natl Cancer Inst 100, 815-825(2008)). Other less common hormone-dependent fusions of ETV1, ETV4, andETV5 could also account for the abiraterone-sensitive cancers (Helgesonet al., Cancer Res 68, 73-80 (2008); Tomlins et al., Science 310,644-648 (2005); Tomlins et al., Cancer Res 66, 3396-3400 (2006)). Besidenovel anti-androgens that target the AR, small molecule inhibitors ofthe phosphatidylinositol-3-kinase (PI3K) are also being evaluated inclinical trials as a strategy for reversing resistance to hormonetherapies (Attard et al., Br J Cancer 95, 767-774 (2006)). Nevertheless,all newly emerging anti-androgen therapies are targeted againstETS-rearrangement positive subset only (30-70% of cases), leaving 10-15%of SPINK1⁻ ETS-negative prostate cancer cases without any effectivetherapy.

Experiments conducted during the course of development of embodiments ofthe present invention demonstrated that targeting SPINK1, either bystable shRNA or an anti-SPINK1 mAb, resulted in decreased tumor growthin 22RV1 (SPINK1⁺/ETS⁻) xenograft models. These results demonstrate boththe importance of identifying molecular subtypes in prostate cancer andtesting potential therapies in appropriate model systems representingsuch molecular subtypes. Indeed, targeting SPINK1 in LNCaP(SPINK1⁻/ETS⁺) or PC3 (SPINK1⁻/ETS⁻) cells, the most commonly used linesfor preclinical testing of therapies for prostate cancer, had no effectin in vitro studies described here and previously (Tomlins et al.,Cancer Cell 13, 519-528 (2008)).

I. Therapeutic Applications

In some embodiments, the present invention provides therapies for cancer(e.g., prostate cancer). In some embodiments, therapies directly orindirectly target SPINK1. In some embodiments, the therapies targetSPINK1 cancer cells that do not harbor an ETS gene fusion (e.g., aTMPRSS2:ETS gene fusion).

In some embodiments, therapies target epidermal growth factor receptor(EGFR), either directly or indirectly. Experiments conducted during thecourse of development of embodiments of the present inventiondemonstrated that co-targeting of both SPINK1 and EGFR resulted inenhanced and additive attenuation of tumor growth. Thus, in someembodiments, a combination of a therapeutic that targets SPINK1 and asecond therapeutic that targets EGFR is utilized.

A. Antibody Therapy

In some embodiments, the present invention provides antibodies thattarget SPINK1 and/or EGFR. Any suitable antibody (e.g., monoclonal,polyclonal, or synthetic) may be utilized in the therapeutic methodsdisclosed herein. In some embodiments, the antibodies described in theexperimental section below are utilized. In some embodiments,commercially available antibodies are utilized (e.g., from Mobitec,Göttingen, Germany). In preferred embodiments, the antibodies used forcancer therapy are humanized antibodies. Methods for humanizingantibodies are well known in the art (See e.g., U.S. Pat. Nos.6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is hereinincorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against SPINK1 and/or EGFR, wherein the antibody is conjugatedto a cytotoxic agent. In such embodiments, a tumor specific therapeuticagent is generated that does not target normal cells, thus reducing manyof the detrimental side effects of traditional chemotherapy. For certainapplications, it is envisioned that the therapeutic agents will bepharmacologic agents that will serve as useful agents for attachment toantibodies, particularly cytotoxic or otherwise anticellular agentshaving the ability to kill or suppress the growth or cell division ofendothelial cells. The present invention contemplates the use of anypharmacologic agent that can be conjugated to an antibody, and deliveredin active form. Exemplary anticellular agents include chemotherapeuticagents, radioisotopes, and cytotoxins. The therapeutic antibodies of thepresent invention may include a variety of cytotoxic moieties, includingbut not limited to, radioactive isotopes (e.g., iodine-131, iodine-123,technicium-99m, indium-111, rhenium-188, rhenium-186, gallium-67,copper-67, yttrium-90, iodine-125 or astatine-211), hormones such as asteroid, antimetabolites such as cytosines (e.g., arabinoside,fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycinC), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), andantitumor alkylating agent such as chlorambucil or melphalan. Otherembodiments include agents such as a coagulant, a cytokine, growthfactor, bacterial endotoxin or the lipid A moiety of bacterialendotoxin. For example, in some embodiments, therapeutic agents includeplant-, fungus- or bacteria-derived toxin, such as an A chain toxins, aribosome inactivating protein, α-sarcin, aspergillin, restrictocin, aribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention justa few examples. In some preferred embodiments, deglycosylated ricin Achain is utilized.

In any event, it is proposed that agents such as these may, if desired,be successfully conjugated to an antibody, in a manner that will allowtheir targeting, internalization, release or presentation to bloodcomponents at the site of the targeted tumor cells as required usingknown conjugation technology (See, e.g., Ghose et al., Methods Enzymol.,93:280 [1983]).

For example, in some embodiments the present invention providesimmunotoxins targeting SPINK1 and/or EGFR. Immunotoxins are conjugatesof a specific targeting agent typically a tumor-directed antibody orfragment, with a cytotoxic agent, such as a toxin moiety. The targetingagent directs the toxin to, and thereby selectively kills, cellscarrying the targeted antigen. In some embodiments, therapeuticantibodies employ crosslinkers that provide high in vivo stability(Thorpe et al., Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solidtumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

In preferred embodiments, antibody based therapeutics are formulated aspharmaceutical compositions as described below. In preferredembodiments, administration of an antibody composition of the presentinvention results in a measurable decrease in cancer (e.g., decrease orelimination of tumor).

B. RNA Interference and Antisense Therapies

In some embodiments, the present invention targets the expression ofSPINK1 and/or EGFR. For example, in some embodiments, the presentinvention employs compositions comprising oligomeric antisense or RNAicompounds, particularly oligonucleotides (e.g., those described herein),for use in modulating the function of nucleic acid molecules encodingSPINK1 and/or EGFR, ultimately modulating the amount of SPINK1 and/orEGFR expressed.

1. RNA Interference (RNAi)

In some embodiments, RNAi is utilized to inhibit SPINK1 and/or EGFRprotein function. RNAi represents an evolutionary conserved cellulardefense for controlling the expression of foreign genes in mosteukaryotes, including humans. RNAi is typically triggered bydouble-stranded RNA (dsRNA) and causes sequence-specific mRNAdegradation of single-stranded target RNAs homologous in response todsRNA. The mediators of mRNA degradation are small interfering RNAduplexes (siRNAs), which are normally produced from long dsRNA byenzymatic cleavage in the cell. siRNAs are generally approximatelytwenty-one nucleotides in length (e.g. 21-23 nucleotides in length), andhave a base-paired structure characterized by two nucleotide3′-overhangs. Following the introduction of a small RNA, or RNAi, intothe cell, it is believed the sequence is delivered to an enzyme complexcalled RISC (RNA-induced silencing complex). RISC recognizes the targetand cleaves it with an endonuclease. It is noted that if larger RNAsequences are delivered to a cell, RNase III enzyme (Dicer) convertslonger dsRNA into 21-23 nt ds siRNA fragments.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001;15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference).

An important factor in the design of siRNAs is the presence ofaccessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem.,2003; 278: 15991-15997; herein incorporated by reference) describe theuse of a type of DNA array called a scanning array to find accessiblesites in mRNAs for designing effective siRNAs. These arrays compriseoligonucleotides ranging in size from monomers to a certain maximum,usually Comers, synthesized using a physical barrier (mask) by stepwiseaddition of each base in the sequence. Thus the arrays represent a fulloligonucleotide complement of a region of the target gene. Hybridizationof the target mRNA to these arrays provides an exhaustive accessibilityprofile of this region of the target mRNA. Such data are useful in thedesign of antisense oligonucleotides (ranging from 7 mers to 25 mers),where it is important to achieve a compromise between oligonucleotidelength and binding affinity, to retain efficacy and target specificity(Sohail et al, Nucleic Acids Res., 2001; 29(10): 2041-2045). Additionalmethods and concerns for selecting siRNAs are described for example, inWO 05054270, WO05038054A1, WO03070966A2, J Mol Biol. 2005 May 13;348(4):883-93, J Mol Biol. 2005 May 13; 348(4):871-81, and Nucleic AcidsRes. 2003 Aug. 1; 31(15):4417-24, each of which is herein incorporatedby reference in its entirety. In addition, software (e.g., the MWGonline siMAX siRNA design tool) is commercially or publicly availablefor use in the selection of siRNAs.

In some embodiments, the present invention utilizes siRNA includingblunt ends (See e.g., US20080200420, herein incorporated by reference inits entirety), overhangs (See e.g., US20080269147A1, herein incorporatedby reference in its entirety), locked nucleic acids (See e.g.,WO2008/006369, WO2008/043753, and WO2008/051306, each of which is hereinincorporated by reference in its entirety). In some embodiments, siRNAsare delivered via gene expression or using bacteria (See e.g., Xiang etal., Nature 24: 6 (2006) and W006066048, each of which is hereinincorporated by reference in its entirety).

In other embodiments, shRNA techniques (See e.g., 20080025958, hereinincorporated by reference in its entirety) are utilized. A small hairpinRNA or short hairpin RNA (shRNA) is a sequence of RNA that makes a tighthairpin turn that can be used to silence gene expression via RNAinterference. shRNA uses a vector introduced into cells and utilizes theU6 promoter to ensure that the shRNA is always expressed. This vector isusually passed on to daughter cells, allowing the gene silencing to beinherited. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC). This complex binds to and cleaves mRNAs which match thesiRNA that is bound to it. shRNA is transcribed by RNA polymerase III.

2. Antisense

In other embodiments, SPINK1 and/or EGFR expression is modulated usingantisense compounds that specifically hybridize with one or more nucleicacids encoding SPINK1 and/or EGFR. The specific hybridization of anoligomeric compound with its target nucleic acid interferes with thenormal function of the nucleic acid. This modulation of function of atarget nucleic acid by compounds that specifically hybridize to it isgenerally referred to as “antisense.” The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity that may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of cancermarkers of the present invention. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. For example,expression may be inhibited to prevent tumor proliferation.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of the present invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. This may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In the present invention, the target is a nucleicacid molecule encoding a SPINK1. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the present invention, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA moleculetranscribed from a gene encoding a tumor antigen of the presentinvention, regardless of the sequence(s) of such codons.

Translation termination codon (or “stop codon”) of a gene may have oneof three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. Similarly, the terms “stop codonregion” and “translation termination codon region” refer to a portion ofsuch an mRNA or gene that encompasses from about 25 to about 50contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon.

The open reading frame (ORF) or “coding region,” which refers to theregion between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the5′ cap site and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5′ cap of an mRNA comprisesan N7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” that are excised from atranscript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites (i.e., intron-exonjunctions) may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition areidentified using commercially available software programs (e.g.,Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India;Antisense Research Group, University of Liverpool, Liverpool, England;GeneTrove, Carlsbad, Calif.). In other embodiments, target sites forantisense inhibition are identified using the accessible site methoddescribed in PCT Publ. No. WO0198537A2, herein incorporated byreference.

Once one or more target sites have been identified, oligonucleotides arechosen that are sufficiently complementary to the target (i.e.,hybridize sufficiently well and with sufficient specificity) to give thedesired effect. For example, in preferred embodiments of the presentinvention, antisense oligonucleotides are targeted to or near the startcodon.

In the context of this invention, “hybridization,” with respect toantisense compositions and methods, means hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases. For example, adenine andthymine are complementary nucleobases that pair through the formation ofhydrogen bonds. It is understood that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds. The antisense compounds in accordance with this inventionpreferably comprise from about 8 to about 30 nucleobases (i.e., fromabout 8 to about 30 linked bases), although both longer and shortersequences may find use with the present invention. Particularlypreferred antisense compounds are antisense oligonucleotides, even morepreferably those comprising from about 12 to about 25 nucleobases.

Chimeric antisense compounds of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the presentinvention as described below.

C. Genetic Therapy

The present invention contemplates the use of any genetic manipulationfor use in modulating the expression of SPINK1 and/or EGFR. Examples ofgenetic manipulation include, but are not limited to, gene knockout(e.g., removing the SPINK1 and/or EGFR gene from the chromosome using,for example, recombination), expression of antisense constructs with orwithout inducible promoters, and the like. Delivery of nucleic acidconstruct to cells in vitro or in vivo may be conducted using anysuitable method. A suitable method is one that introduces the nucleicacid construct into the cell such that the desired event occurs (e.g.,expression of an antisense construct). Genetic therapy may also be usedto deliver siRNA or other interfering molecules that are expressed invivo (e.g., upon stimulation by an inducible promoter (e.g., anandrogen-responsive promoter)).

Introduction of molecules carrying genetic information into cells isachieved by any of various methods including, but not limited to,directed injection of naked DNA constructs, bombardment with goldparticles loaded with said constructs, and macromolecule mediated genetransfer using, for example, liposomes, biopolymers, and the like.Preferred methods use gene delivery vehicles derived from viruses,including, but not limited to, adenoviruses, retroviruses, vacciniaviruses, and adeno-associated viruses. Because of the higher efficiencyas compared to retroviruses, vectors derived from adenoviruses are thepreferred gene delivery vehicles for transferring nucleic acid moleculesinto host cells in vivo. Adenoviral vectors have been shown to providevery efficient in vivo gene transfer into a variety of solid tumors inanimal models and into human solid tumor xenografts in immune-deficientmice. Examples of adenoviral vectors and methods for gene transfer aredescribed in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat.Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106,5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of whichis herein incorporated by reference in its entirety.

Vectors may be administered to subjects in a variety of ways. Forexample, in some embodiments of the present invention, vectors areadministered into tumors or tissue associated with tumors using directinjection. In other embodiments, administration is via the blood orlymphatic circulation (See e.g., PCT publication 99/02685 hereinincorporated by reference in its entirety). Exemplary dose levels ofadenoviral vector are preferably 10⁸ to 10¹¹ vector particles added tothe perfusate.

D. Small Molecule Therapy

Embodiments of the present invention provide small molecules thatinhibit one or more biological activities of SPINK1 and/or EGFR. Smallmolecule therapeutics are identified, for example, using the drugscreening methods described herein.

E. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising pharmaceutical agents that modulate the expression oractivity of SPINK1 and/or EGFR). The pharmaceutical compositions of thepresent invention may be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary(e.g., by inhalation or insufflation of powders or aerosols, includingby nebulizer; intratracheal, intranasal, epidermal and transdermal),oral or parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Other non-antisense chemotherapeutic agents are also withinthe scope of this invention. Two or more combined compounds may be usedtogether or sequentially.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models or based on the examples described herein. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thesubject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

F. Combination Therapy

In some embodiments, the present invention provides therapeutic methodscomprising one or more compositions described herein in combination withan additional agent (e.g., a chemotherapeutic agent). The presentinvention is not limited to a particular chemotherapy agent.

Various classes of antineoplastic (e.g., anticancer) agents arecontemplated for use in certain embodiments of the present invention.Anticancer agents suitable for use with embodiments of the presentinvention include, but are not limited to, agents that induce apoptosis,agents that inhibit adenosine deaminase function, inhibit pyrimidinebiosynthesis, inhibit purine ring biosynthesis, inhibit nucleotideinterconversions, inhibit ribonucleotide reductase, inhibit thymidinemonophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibitDNA synthesis, form adducts with DNA, damage DNA, inhibit DNA repair,intercalate with DNA, deaminate asparagines, inhibit RNA synthesis,inhibit protein synthesis or stability, inhibit microtubule synthesis orfunction, and the like.

In some embodiments, exemplary anticancer agents suitable for use incompositions and methods of embodiments of the present inventioninclude, but are not limited to: 1) alkaloids, including microtubuleinhibitors (e.g., vincristine, vinblastine, and vindesine, etc.),microtubule stabilizers (e.g., paclitaxel (TAXOL), and docetaxel, etc.),and chromatin function inhibitors, including topoisomerase inhibitors,such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide(VM-26), etc.), and agents that target topoisomerase I (e.g.,camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-bindingagents (alkylating agents), including nitrogen mustards (e.g.,mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, andbusulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine,and semustine, etc.), and other alkylating agents (e.g., dacarbazine,hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalentDNA-binding agents (antitumor antibiotics), including nucleic acidinhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines(e.g., daunorubicin (daunomycin, and cerubidine), doxorubicin(adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g.,anthracycline analogues, such as mitoxantrone, etc.), bleomycins(BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4)antimetabolites, including antifolates (e.g., methotrexate, FOLEX, andMEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP,PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir,chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g.,fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine(FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR(ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase,and hydroxyurea, etc.; 6) hormones, including glucocorticoids,antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g.,flutamide, etc.), and aromatase inhibitors (e.g., anastrozole(ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin andcarboplatin, etc.); 8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; 9) biological responsemodifiers (e.g., interferons (e.g., IFN-α, etc.) and interleukins (e.g.,IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growthfactors; 12) agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14)antisense therapy techniques; 15) tumor vaccines; 16) therapies directedagainst tumor metastases (e.g., batimastat, etc.); 17) angiogenesisinhibitors; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors ofacetylation and/or methylation (e.g., HDAC inhibitors); 20) modulatorsof NF kappa B; 21) inhibitors of cell cycle regulation (e.g., CDKinhibitors); 22) modulators of p53 protein function; and 23) radiation.

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of embodiments of the presentinvention. For example, the U.S. Food and Drug Administration maintainsa formulary of oncolytic agents approved for use in the United States.International counterpart agencies to the U.S.F.D.A. maintain similarformularies. The below Table provides a list of exemplary antineoplasticagents approved for use in the U.S. Those skilled in the art willappreciate that the “product labels” required on all U.S. approvedchemotherapeutics describe approved indications, dosing information,toxicity data, and the like, for the exemplary agents.

Aldesleukin Proleukin Chiron Corp., Emeryville, CA (des-alanyl-1,serine-125 human interleukin-2) Alemtuzumab Campath Millennium and ILEX(IgG1κ anti CD52 antibody) Partners, LP, Cambridge, MA AlitretinoinPanretin Ligand Pharmaceuticals, Inc., (9-cis-retinoic acid) San DiegoCA Allopurinol Zyloprim GlaxoSmithKline, Research (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one Triangle Park, NC monosodium salt)Altretamine Hexalen US Bioscience, West(N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine-2,4,6- Conshohocken, PAtriamine) Amifostine Ethyol US Bioscience (ethanethiol,2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester)) AnastrozoleArimidex AstraZeneca Pharmaceuticals,(1,3-Benzenediacetonitrile,a,a,a′,a′-tetramethyl-5-(1H- LP, Wilmington,DE 1,2,4-triazol-1-ylmethyl)) Arsenic trioxide Trisenox CellTherapeutic, Inc., Seattle, WA Asparaginase Elspar Merck & Co., Inc.,(L-asparagine amidohydrolase, type EC-2) Whitehouse Station, NJ BCG LiveTICE BCG Organon Teknika, Corp., (lyophilized preparation of anattenuated strain of Durham, NC Mycobacterium bovis (BacillusCalmette-Gukin [BCG], substrain Montreal) bexarotene capsules TargretinLigand Pharmaceuticals(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- napthalenyl) ethenyl]benzoic acid) bexarotene gel Targretin Ligand Pharmaceuticals BleomycinBlenoxane Bristol-Myers Squibb Co., (cytotoxic glycopeptide antibioticsproduced by NY, NY Streptomyces verticillus; bleomycin A₂ and bleomycinB₂) Capecitabine Xeloda Roche(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine) CarboplatinParaplatin Bristol-Myers Squibb (platinum, diammine[1,1-cyclobutanedicarboxylato(2-)- 0,0′]-,(SP-4-2)) Carmustine BCNU,Bristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea) BiCNUCarmustine with Polifeprosan 20 Implant Gliadel Wafer GuilfordPharmaceuticals, Inc., Baltimore, MD Celecoxib Celebrex SearlePharmaceuticals, (as 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-England pyrazol-1-yl] benzenesulfonamide) Chlorambucil LeukeranGlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)Cisplatin Platinol Bristol-Myers Squibb (PtCl₂H₆N₂) CladribineLeustatin, 2- R. W. Johnson Pharmaceutical(2-chloro-2′-deoxy-b-D-adenosine) CdA Research Institute, Raritan, NJCyclophosphamide Cytoxan, Bristol-Myers Squibb(2-[bis(2-chloroethyl)amino] tetrahydro-2H-13,2- Neosar oxazaphosphorine2-oxide monohydrate) Cytarabine Cytosar-U Pharmacia & Upjohn(1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅) Company cytarabine liposomalDepoCyt Skye Pharmaceuticals, Inc., San Diego, CA Dacarbazine DTIC-DomeBayer AG, Leverkusen,(5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide Germany (DTIC))Dactinomycin, actinomycin D Cosmegen Merck (actinomycin produced byStreptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfa Aranesp Amgen,Inc., Thousand Oaks, (recombinant peptide) CA daunorubicin liposomalDanuoXome Nexstar Pharmaceuticals, Inc.,((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-á-L-lyxo- Boulder, COhexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride) DaunorubicinHCl, daunomycin Cerubidine Wyeth Ayerst, Madison, NJ((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1-naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride)Denileukin diftitox Ontak Seragen, Inc., Hopkinton, MA (recombinantpeptide) Dexrazoxane Zinecard Pharmacia & Upjohn((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- Company piperazinedione)Docetaxel Taxotere Aventis Pharmaceuticals, Inc.,((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, Bridgewater,NJ 13-ester with 5b-20-epoxy-12a,4,7b,10b,13a-hexahydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate) DoxorubicinHCl Adriamycin, Pharmacia & Upjohn(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubex Companyhexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride)doxorubicin Adriamycin Pharmacia & Upjohn PFS Company Intravenousinjection doxorubicin liposomal Doxil Sequus Pharmaceuticals, Inc.,Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly &Company, (17b-Hydroxy-2a-methyl-5a-androstan-3-one propionate)Indianapolis, IN dromostanolone propionate Masterone Syntex, Corp., PaloAlto, CA injection Elliott's B Solution Elliott's B Orphan Medical, IncSolution Epirubicin Ellence Pharmacia & Upjohn((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-arabino- Companyhexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedionehydrochloride) Epoetin alfa Epogen Amgen, Inc (recombinant peptide)Estramustine Emcyt Pharmacia & Upjohn(estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3-[bis(2- Companychloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate, or estradiol 3-[bis(2- chloroethyl)carbamate]17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposidephosphate Etopophos Bristol-Myers Squibb (4′-Demethylepipodophyllotoxin9-[4,6-O-(R)- ethylidene-(beta)-D-glucopyranoside], 4′-(dihydrogenphosphate)) etoposide, VP-16 Vepesid Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia & Upjohn(6-methylenandrosta-1,4-diene-3,17-dione) Company Filgrastim NeupogenAmgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDR Roche(2′-deoxy-5-fluorouridine) Fludarabine Fludara Berlex Laboratories,Inc., (fluorinated nucleotide analog of the antiviral agent CedarKnolls, NJ vidarabine, 9-b-D-arabinofuranosyladenine (ara-A))Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals, Inc.,(5-fluoro-2,4(1H,3H)-pyrimidinedione) Humacao, Puerto Rico FulvestrantFaslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-pentafluoropentylsulphinyl) Guayama, Puerto Ricononyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Gemcitabine Gemzar EliLilly (2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b- isomer))Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33 hP67.6) Goserelinacetate Zoladex AstraZeneca Pharmaceuticals (acetate salt of[D-Ser(But)⁶,Azgly¹⁰]LHRH; pyro-Glu- ImplantHis-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄•(C₂H₄O₂)_(x) Hydroxyurea Hydrea Bristol-Myers SquibbIbritumomab Tiuxetan Zevalin Biogen IDEC, Inc., (immunoconjugateresulting from a thiourea covalent Cambridge MA bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine)Idarubicin Idamycin Pharmacia & Upjohn (5,12-Naphthacenedione,9-acetyl-7-[(3-amino-2,3,6- Companytrideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEXBristol-Myers Squibb(3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) Imatinib Mesilate Gleevec NovartisAG, Basel, (4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-Switzerland (3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamidemethanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La Roche, Inc.,(recombinant peptide) Nutley, NJ Interferon alfa-2b Intron A ScheringAG, Berlin, (recombinant peptide) (Lyophilized Germany Betaseron)Irinotecan HCl Camptosar Pharmacia & Upjohn((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperi- Companydinopiperidino)carbonyloxy]-1H-pyrano[3′,4′: 6,7] indolizino[1,2-b]quinoline-3,14(4H,12H) dione hydrochloride trihydrate) Letrozole FemaraNovartis (4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile)Leucovorin Wellcovorin, Immunex, Corp., Seattle, WA (L-Glutamic acid,N[4[[(2amino-5-formyl-1,4,5,6,7,8- Leucovorinhexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl Ergamisol Janssen Research Foundation,((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo [2,1-b] Titusville, NJthiazole monohydrochloride C₁₁H₁₂N₂S•HCl) Lomustine CeeNU Bristol-MyersSquibb (1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine,nitrogen mustard Mustargen Merck(2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride) Megestrolacetate Megace Bristol-Myers Squibb17α(acetyloxy)-6-methylpregna-4,6-diene-3,20-dione Melphalan, L-PAMAlkeran GlaxoSmithKline (4-[bis(2-chloroethyl) amino]-L-phenylalanine)Mercaptopurine, 6-MP Purinethol GlaxoSmithKline (1,7-dihydro-6H-purine-6-thione monohydrate) Mesna Mesnex Asta Medica (sodium2-mercaptoethane sulfonate) Methotrexate Methotrexate LederleLaboratories (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid) MethoxsalenUvadex Therakos, Inc., Way Exton, Pa(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Mitomycin C MutamycinBristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc., Dublin, CAMitotane Lysodren Bristol-Myers Squibb(1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) MitoxantroneNovantrone Immunex Corporation (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione dihydrochloride)Nandrolone phenpropionate Durabolin-50 Organon, Inc., West Orange, NJNofetumomab Verluma Boehringer Ingelheim Pharma KG, Germany OprelvekinNeumega Genetics Institute, Inc., (IL-11) Alexandria, VA OxaliplatinEloxatin Sanofi Synthelabo, Inc., NY, NY(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] [oxalato(2-)- O,O′] platinum)Paclitaxel TAXOL Bristol-Myers Squibb(5β,20-Epoxy-1,2a,4,7β,10β,13a-hexahydroxytax-11- en-9-one4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia Novartis (phosphonicacid (3-amino-1-hydroxypropylidene) bis-, disodium salt, pentahydrate,(APD)) Pegademase Adagen Enzon Pharmaceuticals, Inc.,((monomethoxypolyethylene glycol succinimidyl) 11-17- (PegademaseBridgewater, NJ adenosine deaminase) Bovine) Pegaspargase Oncaspar Enzon(monomethoxypolyethylene glycol succinimidyl L-asparaginase)Pegfilgrastim Neulasta Amgen, Inc (covalent conjugate of recombinantmethionyl human G- CSF (Filgrastim) and monomethoxypolyethylene glycol)Pentostatin Nipent Parke-Davis Pharmaceutical Co., Rockville, MDPipobroman Vercyte Abbott Laboratories, Abbott Park, IL Plicamycin,Mithramycin Mithracin Pfizer, Inc., NY, NY (antibiotic produced byStreptomyces plicatus) Porfimer sodium Photofrin QLT Phototherapeutics,Inc., Vancouver, Canada Procarbazine Matulane Sigma Tau Pharmaceuticals,(N-isopropyl-μ-(2-methylhydrazino)-p-toluamide Inc., Gaithersburg, MDmonohydrochloride) Quinacrine Atabrine Abbott Labs(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2- methoxyacridine)Rasburicase Elitek Sanofi-Synthelabo, Inc., (recombinant peptide)Rituximab Rituxan Genentech, Inc., South San (recombinant anti-CD20antibody) Francisco, CA Sargramostim Prokine Immunex Corp (recombinantpeptide) Streptozocin Zanosar Pharmacia & Upjohn (streptozocin2-deoxy-2- Company [[(methylnitrosoamino)carbonyl]amino]-a(and b)-D-glucopyranose and 220 mg citric acid anhydrous) Talc Sclerosol Bryan,Corp., Woburn, MA (Mg₃Si₄O₁₀ (OH)₂) Tamoxifen Nolvadex AstraZenecaPharmaceuticals ((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar Schering(3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine- 8-carboxamide)Teniposide, VM-26 Vumon Bristol-Myers Squibb(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac Bristol-MyersSquibb (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien-17-oic acid[dgr]-lactone) Thioguanine, 6-TG Thioguanine GlaxoSmithKline(2-amino-1,7-dihydro-6 H - purine-6-thione) Thiotepa Thioplex ImmunexCorporation (Aziridine,1,1′,1″-phosphinothioylidynetris-, or Tris (1-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin GlaxoSmithKline((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy- 1H-pyrano[3′,4′:6,7] indolizino [1,2-b] quinoline-3,14- 4H,12H)-dione monohydrochloride)Toremifene Fareston Roberts Pharmaceutical Corp.,(2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]-phenoxy)- Eatontown, NJN,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131 TositumomabBexxar Corixa Corp., Seattle, WA (recombinant murine immunotherapeuticmonoclonal IgG_(2a) lambda anti-CD20 antibody (I 131 is aradioimmunotherapeutic antibody)) Trastuzumab Herceptin Genentech, Inc(recombinant monoclonal IgG₁ kappa anti-HER2 antibody) Tretinoin, ATRAVesanoid Roche (all-trans retinoic acid) Uracil Mustard Uracil RobertsLabs Mustard Capsules Valrubicin,N-trifluoroacetyladriamycin-14-valerate Valstar Anthra --> Medeva((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2-oxoethyl pentanoate) Vinblastine, Leurocristine VelbanEli Lilly (C₄₆H₅₆N₄O₁₀•H₂SO₄) Vincristine Oncovin Eli Lilly(C₄₆H₅₆N₄O₁₀•H₂SO₄) Vinorelbine Navelbine GlaxoSmithKline(3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine [R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronicacid Zometa Novartis ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl)phosphonic acid monohydrate)

III. Drug Screening Applications

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for anticancer drugs). The screening methods ofthe present invention utilize cancer markers identified using themethods of the present invention (e.g., including but not limited to,SPINK1 and/or EGFR). For example, in some embodiments, the presentinvention provides methods of screening for compounds that alter (e.g.,decrease) the expression of SPINK1 and/or EGFR. The compounds or agentsmay interfere with transcription, by interacting, for example, with thepromoter region. The compounds or agents may interfere with mRNAproduced from SPINK1 and/or EGFR (e.g., by RNA interference, antisensetechnologies, etc.). The compounds or agents may interfere with pathwaysthat are upstream or downstream of the biological activity of SPINK1and/or EGFR. In some embodiments, candidate compounds are antisense orinterfering RNA agents (e.g., oligonucleotides) directed against cancermarkers. In other embodiments, candidate compounds are antibodies orsmall molecules that specifically bind to SPINK1 and/or EGFR and inhibitits biological function.

In one screening method, candidate compounds are evaluated for theirability to alter SPINK1 and/or EGFR expression by contacting a compoundwith a cell expressing SPINK1 and/or EGFR and then assaying for theeffect of the candidate compounds on expression. In some embodiments,the effect of candidate compounds on expression of SPINK1 and/or EGFR isassayed for by detecting the level of SPINK1 mRNA expressed by the cell.mRNA expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression ofSPINK1 and/or EGFR is assayed by measuring the level of polypeptideencoded by SPINK1 and/or EGFR. The level of polypeptide expressed can bemeasured using any suitable method, including but not limited to, thosedisclosed herein.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., antibodies, proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to SPINK1 and/or EGFR, have aninhibitory (or stimulatory) effect on, for example, SPINK1 and/or EGFRexpression or activity, or have a stimulatory or inhibitory effect on,for example, the expression or activity of a SPINK1 and/or EGFRsubstrate. Compounds thus identified can be used to modulate theactivity of target gene products (e.g., SPINK1 and/or EGFR) eitherdirectly or indirectly in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions. Compounds that inhibit theactivity or expression of cancer markers are useful in the treatment ofproliferative disorders, e.g., cancer, particularly prostate cancer.

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of SPINK1 and/or EGFR protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds that bind to or modulate the activity of a SPINK1 and/orEGFR protein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 Material and Methods Cell Lines and SPINK1 Knockdown

The benign immortalized prostate cell line RWPE, prostate cancer celllines DU145, PC3 and 22RV1 were obtained from the American Type CultureCollection (ATCC, Manassas, Va.) and were grown according to ATCCguidelines. For stable knockdown of SPINK1, human lentiviral shRNAmirindividual clone (ID V2LHS_(—)153419) targeting against SPINK1 ornon-silencing lentiviral shRNAmir in GIPZ vectors were purchased fromOpen Biosystems (Thermo Scientific Open Biosystems, Hunsville, Ala.).Lentiviruses from these constructs were generated by the University ofMichigan Vector Core. Viral particle infections were carried out in thepresence of polybrene (8 μg/ml) in 50-60% confluent 22RV1 cells. After48 hours, infected cells were grown in 22RV1 culture media containingpuromycin (2 μg/ml). Three weeks later, stable cells were plated into 96well plates for clonal selection. SPINK1 knockdown was confirmed inpooled and single clones by qPCR and single clones showing highestknockdown were further expanded. For siRNA mediated knockdowns, the mosteffective siRNA duplex against SPINK1 (J-019724-07; Dharmacon, Chicago)EGFR (J-003114-13; Dharmacon) or siCONTROL Non-Targeting siRNA #1(D-001210-01) was used. All transfections were carried out in thepresence of Oligofectamine (Invitrogen), according to manufacturer'sinstructions. After 24 hr, a second identical transfection was carriedout, and cells were harvested 24 hr later for RNA isolation, invasionassays, or proliferation assays. All transient or stable 22RV1 cellswere tested for SPINK1 knockdown by qPCR. Sequences of siRNAs are asfollows:

J-019724-05: GGAAAUACUUAUCCCAAUG (SEQ ID NO: 5)J-019724-06: UAAUGGAUGCACCAAGAUA (SEQ ID NO: 6)J-019724-07: GAAGAGAGGCCAAAUGUUA (SEQ ID NO: 7)J-003114-13: CAGAGGAUGUUCAAUAACU (SEQ ID NO: 12)

Quantitative PCR (QPCR)

Total RNA was isolated using miRNeasy mini kit following manufacturer'sinstruction (Qiagen). Complimentary DNA was synthesized from onemicrogram of total RNA, using SuperScript III (Invitrogen) in thepresence of random primers. The reaction was carried out for 60 minutesat 50° C. and the cDNA was purified using microcon YM-30 (MilliporeCorp, Bedford, Mass., USA) according to manufacturer's instruction andused as template in quantitative PCRs. All oligonucleotide primers usedin this study were synthesized by Integrated DNA Technologies(Coralville, Iowa). Quantitative PCR (qPCR) was performed using theStepOne Real Time PCR system (Applied Biosystems, Foster City, Calif.).Briefly, reactions were performed with SYBR Green Master Mix (AppliedBiosystems) and 25 ng of both the forward and reverse primers for SPINK1(TGTCTGTGGGACTGATGGAA (SEQ ID NO:1)) and AGGCCCAGATTTTTGAATGA (SEQ IDNO:2), PRSS1 (5′-GCCTGGACGCTCCTGTGCTG-3 (SEQ ID NO:8)′ and5′-CTGGGCACAGCCATCACCCC-3′ (SEQ ID NO:9)) and EGFR(5′-GGGCCAGGTCTTGAAGGCTGT-3′ (SEQ ID NO:10) and5′-ATCCCCAGGGCCACCACCAG-3′ (SEQ ID NO:11)) using the manufacturerrecommended thermocycling conditions. For each experiment, thresholdlevels were set during the exponential phase of the qPCR reaction usingthe StepOne software. The amount of each target gene relative to thehousekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH;forward TGCACCACCAACTGCTTAGC (SEQ ID NO:3) and reverse primersGGCATGGACTGTGGTCATGAG (SEQ ID NO:4)) for each sample was determinedusing the comparative threshold cycle (Ct) method. Threshold levels foreach experiment were set during the exponential phase of the QPCRreaction using Sequence Detection Software version 1.2.2 (AppliedBiosystems).

Cell Proliferation Assay

Proliferation for control and experimental cells was measured by acolorimetric assay based on the cleavage of the tetrazolium salt WST-1by mitochondrial dehydrogenases (cell proliferation reagent WST1; RocheDiagnostics, Mannheim, Germany) at the indicated time points intriplicate. Cell counts for shNS vector and shSPINK1 cells wereestimated by trypsinizing cells and analysis by Coulter counter (BeckmanCoulter, Fullerton, Calif., USA) at different time points intriplicates.

Soft Agar Colony Assay

A 50 μL base layer of agar (0.6% Agar in DMEM with 10% FBS) was allowedto solidify in a 96-well flat-bottom plate prior to the addition of 754,stable shNS vector and shSPINK1 cell suspension containing 4,000 cellsin 0.4% Agar in DMEM with 10% FBS. The cell containing layer was thensolidified at 4° C. for 15 minutes prior to the addition of 1004, of MEMwith 5% FBS. Colonies were allowed to grow for 21 days before imagingunder a light microscope.

Cell Motility Assay

22RV1 or stable shNS vector and shSPINK1 cells were plated on a lawn ofmicroscopic fluorescent beads on collagen coated 96-well plates(Cellomics, Thermo Scientific, Pa., US). Motile cells push the beads andcreate phagokinetic tracks behind each cell. The cleared track area isproportional to the magnitude of cell motility. Plates were analyzed andimages were captured using standard light microscopy.

Basement Membrane Matrix Invasion Assay

For invasion assays, shNS vector or shSPINK1 cells, RWPE, PC3 and 22RV1cells were used. Equal numbers of the indicated cells were seeded ontothe basement membrane matrix (EC matrix; Chemicon, Temecula, Calif.,USA) present in the insert of a 24-well culture plate, with fetal bovineserum added to the lower chamber as a chemoattractant. After 48 hr,non-invading cells and EC matrix were removed using a cotton swab.Invaded cells were stained with crystal violet and photographed. Theinserts were treated with 10% acetic acid, and absorbance was measuredat 560 nm.

22RV1 or SPINK1 Knockdown Xenograft Models

Four weeks old male Balb/C nu/nu mice were purchased from Charles River,Inc. (Charles River Laboratory, Wilmington, Mass.). StableshNS-luciferase and shSPINK1-luciferase cells (5×10⁵ cells) or 22RV1-Luc(2×10⁵ cells) or PC-3 luciferase cells were resuspended in 100 μl ofsaline with 20% Matrigel (BD Biosciences, Becton Drive, N.J.) and wereimplanted subcutaneously into the left flank regions of the mice. Micewere anesthetized using a cocktail of xylazine (80-120 mg/kg IP) andketamine (10 mg/kg IP) for chemical restraint before implantation. Eightmice were included in each group. Mice implanted with 22RV1-luciferaseor PC-3 luciferase cells were randomly divided into two groups, andtreated twice a week with SPINK1 mAb (Mobitec Inc; Goettingen, Germany)or control mouse IgG antibody (Millipore; Kankakee, Ill.) at the dose of10 mg/Kg body weight. Epitope mapping of the SPINK1 mAb is shown in FIG.17. Growth in tumor volume was recorded weekly by using digital calipersand tumor volumes were calculated using the formula (π/6) (L×W2), whereL=length of tumor and W=width. Antitumor activity was determined fromthe analyses of tumor growth inhibition, defined as the decrease in themean tumor volume for SPINK1 mAb treated mice versus mouse IgG mAbtreated mice. In vivo bioluminescent imaging was performed weekly uptoweek 4 using IVIS-200 imaging system (Xenogen Corp.). Mice were injected150 mg/kg luciferin intra-peritoneal 12 min before imaging. All imageswere collected and analyzed with Living Image software (Xenogen Corp.).All procedures involving mice were approved by the University Committeeon Use and Care of Animals (UCUCA) at the University of Michigan andconform to their relevant regulatory standards.

Immunohistochemistry and Immunofluorescence Staining

FFPE sections were obtained from formalin-fixed xenografted tumors and,antigen retrieval was performed by microwaving sections in citratebuffer (pH6.0) for 10 min, followed by cooling and rinse in water.Sections were further blocked in hydrogen peroxidase for 5 min followedby incubation in anti-Ki-67 antibody at 1:400 dilutions (AbCam,Cat#ab15580) for 30 min at room temperature. Sections were furtherincubated in EnVision+ for 30 min at room temperature; immunoreactivenuclei were visualized with the Vectastain Elite ABC Kit usingdiaminobenzidine (DAB) as the substrate (Vector Laboratories, Inc.).Finally, sections were counterstained with Harris Hematoxlyin (Fisher),dehydrated, and mounted with Permount (Fisher). Immunoreactive positiveKi-67 nuclei were scored blindly for both groups in 400× magnification.

For immunofluorescence staining, shNS-luciferase and shSPINK1-luciferasecells were grown in chamber slides at sub-confluent density. Cells werefixed using chilled methanol after washing with 1XPBS. The chamberslides containing cells were then blocked in PBS-T containing 5% normaldonkey serum for 1 hour at room temperature. Slides were incubatedovernight at 4° C. with a mouse anti-SPINK1 antibody (H00006690-M01;Abnova, Taipei City, Taiwan) 1:10,000 dilution, then washed, andfollowed by secondary antibodies (anti-mouse Alexa 555 1:1000 dilutions)for 1 hour. Slides were mounted using Vectashield mounting mediumcontaining DAPI (Vector Laboratories, Burlingame, Calif.) after washingwith PBS-T and PBS. Fluorescence images were captured using a ZeissMicroscope (Carl Zeiss, Gottingen, Germany) equipped with a highresolution CCD camera controlled by ISIS image processing software(Metasystems, Germany).

Statistical Analysis

All values presented in the study were expressed as mean±SEM. Thesignificant differences between the groups were analyzed by a Student'st test and a P value of <0.05 was considered significant.

Production of Recombinant SPINK1 Protein

A synthetic construct with SPINK1-2xV5 cloned into pcDNA3.1 using KpnIand XhoI restriction sites was purchased from GENEART (Regensburg,Germany). The full length SPINK1 cDNA including signal peptide wasamplified and subcloned downstream of the Met-Arg-Gly-Ser-His₆ (MRGSH₆)tag coding sequence into the BamHI/PstI restriction enzyme sites of thepQE-9 expression vector (Qiagen, Hilden, Germany). The plasmid wassequenced to verify integrity. Production of the recombinant proteinfrom pQE-9 plasmids was carried out using M15 cells treated withisopropyl-1-thio-D-galactopyranoside (400 mM). Bacterial cell lysateswere centrifuged and the supernatants were purified by affinitychromatography using a Co²⁺-agarose resin (Clontech,Saint-Germain-en-Laye, France). Multiple tags protein including 6× Hisprotein (GenScript Corp., Piscataway, N.J.) was used as a control.Bacterially expressed recombinant SPINK1 protein was separated on 5-30%sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. Inorder to get concentrated SPINK1 fraction from CM of 22RV1 cells orcontrol CM form RWPE cells, proteins were separated by ultrafiltrationaccording to their molecular weight (MW), using membranes at a cutoff of3-10 kDa (SPINK1 molecular weight 6.2 kDa). Protein was transferred ontoPolyvinylidene Difluoride membrane (GE Healthcare) and membrane wasincubated for one hour in blocking buffer [Tris-buffered saline, 0.1%Tween (TBS-T), 5% nonfat dry milk] and probed with the SPINK1 mAb(Mobitec Inc; Goettingen, Germany). After washing the blots with TBS-T,the blots were incubated with horseradish peroxidase-conjugatedsecondary mouse antibody and the signals visualized by enhancedchemiluminescence system as described by the manufacturer (GEHealthcare).

Immunoprecipitation and Western Blot Analysis

Briefly, transiently EGFR expressing HEK293 cells were washed twice PBSsupplemented with protease inhibitor. Cells were lysed in Triton X-100lysis buffer (20 mM MOPS, pH 7.0, 2 mM EGTA, 5 mM EDTA, 30 mM sodiumfluoride, 60 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mMsodium orthovanadate, 1% Triton X-100, protease inhibitor cocktail(Roche). Cell lysates (0.5-1.0 mg) were then pre-cleaned with proteinA/G agarose beads (Santa Cruz) by incubation for 1 hour with shaking atroom temperature followed by centrifugation at 2000× g for 1 minute.Recombinant SPINK1-GST (Proteintech Group Inc.), GST (AbCam) or GST-VEGFReceptor 2 protein (Cell Signaling) (80 μg/ml) were added to thepre-cleaned protein lysates and incubated at 4° C. overnight. Similarly,22Rv1 cells lysate in Kinet lysis buffer was mixed with SPINK1-GSTrecombinant protein (80 m/ml) and incubated at 4° C. overnight. Afteradding 2 μg of each antibody (mouse IgG, Millipore; SPINK1 mAb, MobitecInc.; EGFR mAb, Cell Signaling) lysates were further incubated withshaking at 4° C. for 4 hours prior to addition of 20 μL protein A/Gagarose beads (Santa Cruz). The mixture was then incubated with shakingat 4° C. for another 4 hours prior to washing the lysate-beadprecipitate (centrifugation at 2000×g for 1 minute) 3 times in TritonX-100 lysis buffer or Kinet lysis buffer.

Beads were finally precipitated by centrifugation, resuspended in 254,of 2× loading buffer and boiled at 80° C. for 10 minutes. Samples werethen analyzed by SDS-PAGE Western blot analysis as described below.

Western Blot Analysis

Cell lysates were prepared in RIPA lysis buffer (Thermo Scientific),supplemented with complete proteinase inhibitor and phosphataseinhibitor mixture (Roche). Fifteen micrograms of each protein extractwas boiled in sample buffer, separated by SDS-PAGE, and transferred ontoPolyvinylidene Difluoride membrane (GE Healthcare). rSPINK1 stimulated22RV1 phospho-EGFR blot was performed as described before (Jemal et al.,2010. CA Cancer J Clin 60, 277-300 (2010)). The membrane was incubatedfor one hour in blocking buffer (Tris-buffered saline, 0.1% Tween(TBS-T), 5% nonfat dry milk) and incubated overnight at 4° C. withanti-phospho-MEK or -ERK or -EGFR or -AKT antibodies or total -MEK or-ERK or -AKT and -EGFR antibodies (Cell Signaling); trypsinl polyclonalantibody (Abcam) and PSA monoclonal antibody (Dako). Following threewashes with TBS-T, the blot was incubated with horseradishperoxidase-conjugated secondary antibody and the signals visualized byenhanced chemiluminescence system as described by the manufacturer (GEHealthcare).

EGFR Cross Linking Immunoblotting

22RV1 cells were stimulated with rSPINK1 (100 ng/ml) or EGF (10 ng/ml)in 6-well plates for 0, 5, 10, 30, 60 and 90 min at 37° C. At the end ofeach time point cells were washed twice with 1× PBS. Cells were thentreated with cross linking reagent BS3 [Bis-(sulfosuccinimidyl)suberate] to a final concentration of 5 mM and incubated on ice for 2 h.The quench solution (1M Tris, pH 7.5, 1:100 dilutions) was then added toa final concentration of 10 mM and incubated for 15 min on ice. Thecells were then lysed with RIPA buffer supplemented with proteaseinhibitor and phosphatase inhibitors (Roche). EGFR dimerization wasanalyzed by Non-reducing immunoblot.

Serum Toxicity Marker Analyses

At the end of the xenograft study, mice were anaesthetized and blood wascollected by cardiac puncture. Blood was transferred into a 1.5 mleppendorf tube and kept on ice for 45 min, followed by centrifugation at8000 rpm for 10 min at 4° C. Clear supernatant containing serum wascollected and transferred into a sterile 1.5 ml eppendorf tube. Allserum markers were measured using dry-slide technology on IDEXX Vettest8008 biochemical analyser (IDEXX, France). About 50 μL of the serumsample was loaded on the VetTest pipette tip followed by securelyfitting it on the pipettor and manufacturer's instructions were followedfor further analyses.

Chick Chorioallantoic Membrane (CAM) Assay

The assay was performed essentially as described (Zijlstra et al.,Cancer Res 62, 7083-7092 (2002)). Two million RWPE cells were mixed witheither 200 ng multiple tag control protein or 200 ng of rSPINK1 proteinand applied to the chorioallantoic membrane (CAM) of 11-day old chickenembryo. Similarly, two million 22RV1 or PC3 cells were mixed with eithermonoclonal IgG or anti-SPINK1 or C225 (1 μg/ml) and applied onto theupper CAM of a fertilized chicken embryo. Three days post-implantation,the relative number of cells that intravasate into the vasculature ofthe lower CAM was analyzed by extracting genomic DNA using the PurgeneDNA purification system. Quantification of the human cells in theextracted DNA was done as described (van der Horst et al., Biotechniques37, 940-942, 944, 946 (2004)).

Results SPINK1 as an Autocrine Factor in Prostate Cancer

This Example describes the investigation of the role of autocrine SPINK1in invasion and proliferation using recombinant 6× His-tagged SPINK1protein (rSPINK1) (FIG. 5) or conditioned media (CM) collected from22RV1 prostae cancer cells. Benign immortalized RWPE prostate epithelialcells and DU145 and PC3 prostate cancer cells (both SPINK1⁻/ETS⁻) weretreated with rSPINK1 (10 ng/ml). This resulted in a significant increasein cell proliferation compared to controls over a six day time course(FIG. 1A). The effect of rSPINK1 or CM on cell invasion was nextcharacterized using a Boyden chamber Matrigel invasion assay. As shownin FIG. 1B, addition of rSPINK1 or CM to RWPE cells significantlyincreased invasion (P=0.003, P=0.0009 respectively), which wasattenuated (P=0.008) by neutralizing with SPINK1 mAb. Similar effectswere observed when a breast cancer MCF7 cells were treated with rSPINK1or CM either in the presence or absence of anti-SPINK1 mAb (FIG. 6). CMfrom RWPE cells or multiple 6× His tagged protein controls did notmediate invasion of RWPE or 22RV1 cells (FIG. 7).

It was previously shown that transient siRNA mediated silencing ofSPINK1 in 22RV 1 cells decreased cell invasion (Tomlins et al, CancerCell 13, 519-528 (2008)). In this study, several siRNA sequences hadsimilar phenotypic effects and the one with the most robust knock-downwas identified for subsequent studies. These results were extended bydemonstrating that the addition of rSPINK1 or 22RV1 CM to 22RV1 cellstreated with siRNA against SPINK1 rescued the invasive phenotype (FIG.1C, P=0.001 in both cases). Together, these findings show thatextracellular SPINK1 induces prostate cancer cell proliferation andinvasion, indicating that SPINK1 is an autocrine pro-proliferative andpro-invasive factor.

It was next investigated whether the exogenous effect of SPINK1 on cellproliferation and invasion is dependent on protease inhibitory activityof trypsin (which has been shown to be simultaneously expressed withSPINK1 in different tumor types (Hotakainen et al., Int J Oncol 28,95-101 (2006); Paju et al., Clin Cancer Res 10, 4761-4768 (2004)) orPSA. Experiments demonstrated that PRSS1 (trypsinogen) mRNA expressionin 22RV1 cells is relatively low (FIG. 14A), although a significantincrease in PRSS1 transcript was observed in siRNA mediated SPINK1knockdown 22RV1 cells (FIG. 14B). However, as shown in FIG. 14C,stimulation of 22RV1 cells with rSPINK1 or EGF did not affect trypsinexpression. siRNA mediated knockdown of PRSS1 in 22RV1 cells also had noeffect on invasion (FIGS. 14D & E). Similarly, stimulation of 22RV1cells with rSPINK1 or EGF did not affect PSA expression (FIG. 15A).Finally, blocking PSA using a monoclonal antibody did not significantlyinhibit 22RV1 cell invasion (FIG. 15B). Together, these findingsdemonstrate that extracellular SPINK1 induces prostate cancer cellproliferation and invasion independent of protease inhibitory activityof trypsin or PSA. The results indicate that SPINK1 is an autocrinepro-proliferative and pro-invasive factor with effects independent ofprotease activity.

Role of SPINK1 in Cell Proliferation and Invasion

To further investigate the role of SPINK1 in cell proliferation andinvasion, shRNA against SPINK1 were generated and stable 22RV1 cells inwhich SPINK1 was silenced (shSPINK1) were established. Knockdown ofSPINK1 in both pooled and clonal shSPINK1 cells was confirmed at the RNAlevel by quantitative PCR and at the protein level by immunoflourescencestaining using an antibody against SPINK1 (FIG. 2A). Next, the effect ofSPINK1 on cell invasion and motility was investigated using shSPINK1cells. shSPINK1 cells showed decreased invasion in a Boyden chamberMatrigel assay compared to non-specific vector control (shNS) cells(FIG. 2B; P=0.002 in both cases). shSPINK1 cells also showed reducedcell motility compared to shNS cells in a bead motility assay (FIG. 2B).

To investigate the role of SPINK1 in cell proliferation, assays wereperformed using pooled shSPINK1 cells, the clone with the greatestSPINK1 knockdown (shSPINK1 clone 11), and 22RV1 cells with stableknockdown of a non-specific vector control (shNS). Both pooled andsingle clone of shSPINK1 cells showed a significantly decreasedproliferation compared to shNS cells (FIG. 2C; P=0.00002 in both cases).By soft agar colony formation assay, shSPINK1 cells also showeddecreased colonies compared to shNS vector cells (FIG. 2D).

In Vitro Targeting of SPINK1 Using a Monoclonal Antibody

As a monoclonal antibody was able to attenuate the increased cellinvasion caused by rSPINK1 in RWPE cells (FIG. 1B), it was contemplatedthat this antibody may be used to directly target SPINK1⁺/ETS⁻ prostatecancer cells. Thus, the effects of the anti-SPINK1 mAb (Mobitec,Gottingen, Germany) on 22RV1 cell invasion were assayed, and it wasfound that the anti-SPINK1 mAb (0.5-1 μg/ml) significantly attenuatedcell invasion as compared to a control monoclonal IgG antibody (FIG. 3A;P=0.0003 and P=0.0007 respectively). The anti-SPINK1 mAB had nosignificant effect on PC3 (SPINK1⁻/ETS⁻) prostate cancer cell invasion(FIG. 3B). Similarly, the anti-SPINK1 mAb attenuated 22RV1 cell motilitycompared to IgG control, but had no effect on DU145 or PC3 cell motility(FIG. 8).

In addition to inhibiting proliferation, anti-SPINK1 mAb (0.5 and 1μg/ml) significantly attenuated cell invasion by 69% and 81%respectively as compared to a control IgG mAb in 22RV1 cells (FIG. 3C;P=0.002 and P=0.007 respectively). Similar to 22RV1, which is anandrogen signaling independent derivative of primary CWR22 humanprostate xenograft tumors. CWR22Pc cells, an androgen signalingdependent derivative of CWR22 (Dagvadorj et al., Clin Cancer Res 14,6062-6072 (2008)), which also express high levels of SPINK1, were alsoinvestigaed. CWR22Pc cell invasion was blocked by 47 and 54% byanti-SPINK1 mAb at 0.5 and l μg/ml of SPINK1 mAb concentration (FIG. 3C;P=0.003 and P=0.002 respectively). The anti-SPINK1 mAb had nosignificant effect on invasion of SPINK1-prostate cancer cell linesincluding PC3, DU145, LNCaP or VCaP (FIG. 3C). The anti-SPINK1 mAbattenuated 22RV1 cell motility compared to IgG control, but had noeffect on PC3 (SPINK1−/ETS−) cell motility (FIG. 8A).

Oncogenic Effects of SPINK1 in Part Through Interaction with EGFR

SPINK1 has a similar structure as epidermal growth factor (EGF), withapproximately 50% sequence homology and three intrachain disulfidebridges (Hunt et al., Biochem Biophys Res Commun 60, 1020-1028 (1974);Bartelt et al., Arch Biochem Biophys 179, 189-199 (1977)). Tocharacterize potential SPINK1 and EGFR interaction, EGFR wasoverexpressed EGFR in human embryonic kidney cells (HEK) 293 cells. Thelysates were incubated with SPINK1-GST, GST or GST-VEGF Receptor 2(GST-VEGFR) recombinant proteins. A strong interaction betweenSPINK1-GST and EGFR was observed but not with GST alone or GST-VEGFRrecombinant protein (FIG. 11A; top panel).

Endogenous SPINK1 and EGFR interaction was not detected byimmunoprecipitation and immunoblotting in 22RV1 cells, due to secretorynature of the SPINK1 protein. Addition of GST-SPINK1 to 22RV1 cellsfollowed by immunoprecipitation and immunoblotting confirmed theinteraction of SPINK1 and endogenous EGFR in 22RV1 cells (FIG. 11A;bottom panel).

To further delineate the role of EGFR mediation of SPINK1 in prostatecancer, it was next assessed whether exogenous SPINK1 was capable ofinducing EGFR phosphorylation (similar to the cognate ligand EGF).Stimulating 22RV1 cells with rSPINK1 resulted in EGFR phosphorylation,although weaker than that observed with EGF (FIG. 11B). rSPINK1stimulation resulted in sustained EGFR phosphorylation over a 90 minutetime course, while EGF resulted in strong EGFR phosphorylation whichdiminished after only 10 min. Similarly, stable shSPINKJ knockdown 22RV1cells (pooled and clonal) showed decreased phosphorylated EGFR (pEGFR),with slightly decreased total EGFR (FIG. 16A). It was also demonstratedthat rSPINK1 is able to induce dimerization of EGFR, although moreweakly than EGF (FIG. 16B).

The functional consequences of SPINK1-EGFR interaction in the context ofSPINK1+ prostate cancer was examined using 22RV1 cells. Transientknockdown of EGFR (FIG. 8B) blocked 22RV1 cell invasion by 75% (FIG.11C; P=0.004) which was partially rescued by addition of exogenousSPINK1. A similar effect of EGFR knockdown was observed in RWPE cellstreated with recombinant SPINK1 (FIG. 11D; P=0.014 and P=0.021respectively). These results indicate that some of SPINK1's effects aremediated by EGFR.

It was next determined whether EGFR blockade could inhibit the oncogeniceffects of SPINK1. It was first demonstrated that mAb to EGFR(cetuximab, C225) blocked the cell invasive effects of rSPINK1 in RWPEcells (FIG. 11E). C225 also blocked cell invasion of SPINK1+ 22RV1 cellsbut not in SPINK1-cell lines DU145, PC3, LNCaP or VCaP (FIG. 11F).Combining mAbs to SPINK1 and EGFR had an additive effect in theinhibition of 22RV1 cell invasion (FIG. 11G; P=0.001). In contrast tomAb to SPINK1 (FIG. 11A), C225 had no effect on 22RV1 cell proliferationor PC3 and DU145 cells proliferation (FIG. 11H). Together, theseexperiments indicate that SPINK1 has both EGFR-dependent andEGFR-independent functions in prostate cancer.

To investigate the downstream signaling pathways involved in theSPINK1-EGFR axis, the the mitogen-activated protein kinase (MAPK) andprotein kinase B/AKT pathways were investigated in stable SPINK1knockdown 22RV1 cells (shSPINK1 clone 11). Decreased pMEK, pERK and pAKTin stable shSPINK1 cells compared to control shNS cells was observed(FIG. 8C). Likewise, 22RV1 cells treated with SPINK1 mAb antibody showeddecreased pERK (FIG. 8D).

In Vivo Targeting of SPINK1

The in vitro studies showed that SPINK1 mediates cell proliferation andinvasion in SPINK1⁺ prostate cancer cells. An in vivo model was used toassay a mAb for targeting SPINK1⁺ cancer cells in vivo. To qualifySPINK1 as a therapeutic target, shSPINK1-luciferase and shNS-luciferasevector cells were implanted in nude mice. At both 4 and 5 weeks22RV1-shSPINK1-luciferase cells formed significantly smaller tumors (55%reduction at week 4; P=0.013 and 63% reduction at week 5; P=0.008)compared to shNS-luc vector cells (FIG. 4A). This effect is especiallydramatic considering that a pooled population of shSPINK1 cells (and nota clone) was used.

To demonstrate the preclinical efficacy of the anti-SPINK1 mAb, nudemice implanted with 22RV1-luciferase cells were treated with SPINK1 mAb(10 mg/kg body weight) or mouse monoclonal IgG (10 mg/kg body weight)twice a week. As shown in FIG. 4B, administration of SPINK1 mAb resultedin a 59% reduction of tumor burden at week 4 (P=0.015) and 55% reductionat week 5 (P=0.015). Similarly, there was a 60% reduction in tumorburden at week 4 as assessed by bioluminescence imaging. A significantdecrease in Ki-67 positive nuclei and mitoses were recorded in theSPINK1 mAb treated group as compared to the control group (FIG. 9).There was no evidence of morbidity in either group, weekly body weightswere similar in both groups, and there was no difference in mean amylaseor lipase between treated and control groups (FIG. 10).

As SPINK1 mediates its oncogenic effects in part through EGFR, the mAbto EGFR (C225) was assessed using the same dosage schedule. C225treatment resulted in a 41% reduction at week 4 (P=0.04) and 37%reduction at week 5 (P=0.02) (FIGS. 4E & I). By combining mAbs to SPINK1and EGFR an additive effect was observed in vivo showing a 74% and 73%reduction in the growth of 22RV1 xenografts at week 4 (P=0.01) and 5(P=0.003) respectively (FIGS. 4F & I). To confirm the in vitro results,which indicate no effect of SPINK1 or EGFR inhibition on SPINK1-prostatecancer, a xenograft study was performed using PC3 cells. Neither SPINK1mAb nor C225 significantly inhibited tumor growth in PC3 xenograftedmice (FIGS. 4G & 4I).

To investigate the role of SPINK1 in intravasation, a chickchorioallantoic membrane (CAM) model system was used (Zijlstra et al.,Cancer Res 62, 7083-7092 (2002)) to demonstrate that rSPINK1 inducedintravasation of benign RWPE cells (FIG. 4A). SPINK1 mAb and C225significantly inhibit 22RV1 cell intravasation (P=0.01 and P=0.03respectively), but did not significantly inhibit PC3 cell intravasation(FIGS. 4B & C).

All publications, patents, patent applications and accession numbersmentioned in the above specification are herein incorporated byreference in their entirety. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications and variations of thedescribed compositions and methods of the invention will be apparent tothose of ordinary skill in the art and are intended to be within thescope of the following claims.

1. A method of inhibiting at least one biological function of a serinepeptidase inhibitor, Kazal type I (SPINK1) polypeptide, comprisingcontacting said SPINK1 polypeptide with an antibody that specificallybinds to said SPINK1 polypeptide and inhibits at least one biologicalfunction of said SPINK1 polypeptide.
 2. The method of claim 1, whereinsaid SPINK1 is secreted by a cell.
 3. The method of claim 2, whereinsaid cell is a cancer cell.
 4. The method of claim 3, wherein saidcancer cell is a prostate cancer cell.
 5. The method of claim 2, whereinsaid cell is in vivo.
 6. The method of claim 5, wherein said cell is inan animal.
 7. The method of claim 6, wherein said animal is a human. 8.The method of claim 2, wherein said cell is ex vivo.
 9. The method ofclaim 2, wherein said inhibiting at least one biological function ofSPINK1 inhibits the proliferation of said cell.
 10. The method of claim2, wherein said inhibiting at least one biological function of SPINK1inhibits the invasiveness of said cell.
 11. The method of claim 2,further comprising the step of administering a second agent to saidcell.
 12. The method of claim 11, wherein said second agent inhibits atleast one biological function of EGFR.
 13. The method of claim 11,wherein said second agent is an anti-cancer therapeutic agent.
 14. Themethod of claim 2, wherein said cell does not harbor an ETS gene fusion.15. The method of claim 14, wherein said ETS gene fusion is aTMPRSS2:ETS gene fusion.
 16. A kit, comprising a pharmaceuticalcomposition that inhibits at least one biological function of SPINK1,wherein said composition comprises an antibody that specifically bindsto SPINK1 and inhibits at least one biological function of SPINK1. 17.The kit of claim 16, further comprising a reagent that inhibits at leastone biological function of EGFR.